Components of the Human Gastrointestinal Tract
The gastrointestinal tract is a continuous tube that extends from the mouth to the anus. On a gross level, the gastrointestinal tract is composed of the following organs: the mouth, most of the pharynx, the esophagus, the stomach, the small intestine (duodenum, jejunum and ileum), and the large intestine (cecum including the appendix, colon and rectum) (Tortora, G. J., et al., Principles of Anatomy and Physiology, Wiley & Sons, Inc. Hoboken, N.J. 2006). Each segment of the gastrointestinal tract participates in the absorptive processes essential to digestion by producing chemical substances that facilitate digestion of foods, liquids, and other substances, such as therapeutic agents, taken orally.
Within the gastrointestinal tract, the small intestine is the site of most digestion and absorption and is structured specifically for these important functions. The small intestine is divided into three segments: the duodenum, the jejunum, and the ileum. The absorptive cells of the small intestine produce several digestive enzymes known as ‘brush-border’ enzymes. The brush-border enzymes, together with pancreatic and intestinal juices, facilitate the absorption of substances from the chime in the small intestine. The large intestine is the terminal portion of the gastrointestinal tract and contributes to the completion of absorption, the production of certain vitamins, and the formation and expulsion of feces.
The epithelium is a purely cellular avascular tissue layer that covers all free surfaces (cutaneous, mucous, and serous) of the body, including the glands and other structures derived from it. The epithelium lines both the exterior of the body, as skin, the interior cavities, and lumen of the body. The outermost layer of human skin is composed of dead stratified squamous, keratinized epithelial cells. The mucous membranes lining the inside of the mouth, the esophagus, and parts of the rectum are lined by nonkeratinized stratified squamous epithelium. Epithelial cell lines also are present inside of the lungs, the gastrointestinal tract, and the reproductive and urinary tracts, and form the exocrine and endrocrine glands.
Epithelial cells are involved in secretion, absorption, protection, transcellular transport, sensation detection and selective permeability. There are variations in the cellular structures and functions in the epithelium throughout the gastrointestinal tract. The epithelium in the mouth, pharynx, esophagus and anal canal is mainly a protective, nonkeratinized, squamous epithelium that has a protective role against abrasion and wear-and-tear from mechanical movements of food particles that are chewed and swallowed (mouth, pharynx, and esophagus) or undigested/unabsorbed substances eliminated by defecation (anal canal). The epithelium of the stomach is composed of several kinds of cells. Columnar cells, present in a single layer, participate minimally in absorption and secretion. Goblet cells produce mucus and participate in protective and mechanical functions. Enteroendocrine cells participate in the secretion of gastrointestinal hormones. Additionally, the columnar epithelial lining of the stomach has millions of gastric pits, which lead to gastric glands. Various types of specialized epithelial cells located in these gastric glands produce gastric juice that contains pepsin (an enzyme produced by Chief cells and needed for protein digestion), intrinsic factor (needed for absorption of vitamin B12) and hydrochloric acid to decontaminate food and activate pepsin) produced by Parietal cells, plus mucus (produced by surface mucous cells and mucous neck cells) in the stomach. Simple columnar epithelia, with microvilli, line the lumen to facilitate the role of the small intestine as the primary absorptive organ in the body; as the absorptive function of the large intestine is not as great as that of the small intestine, microvilli are not present in epithelia of the large intestine. Within the large intestine, protective mucus is produced in copious amounts and goblet cells are abundant. The epithelial lining provides an important defense barrier against microbial pathogens throughout the GI tract.
The development of intestinal epithelium involves three major phases: 1) an early phase of epithelial proliferation and morphogenesis; 2) an intermediate period of cellular differentiation in which the distinctive cell types characteristic of intestinal epithelium appear; and 3) a final phase of biochemical and functional maturation (Mathan, M. P., et al. Am. J. Anat. 146(1):73-92. 1976; Hirano, S. and Kataoka, K. Arch. Histol. Jpn. 49(3):333-48. 1986; Bjerknes, M. and Cheng, H. Am. J. Physiol. Gastrointest. Liver Physiol. 283(3):G767-77. 2002; Brittan, M. and Wright, N. A. J. Pathol. 197(4):492-5. 2002; Potten, C. et al. Cell Prolif. 36(3):115-29. 2003; Sancho, E., et al. Curr. Opin. Cell. Biol. 15(6):763-70. 2003; Stappenbeck, T., et al. Proc. Natl. Acad. Sci. USA. 100(3):1004-9. 2003).
Intestinal crypts, located at the base of villi, contain stem cells (Burgess, D., et al. J. Cell Biol. 109(5):2139-44. 1989; Weiser, M., et al., Immnunol. Invest. 18(1-4):417-30. 1989; Bjerknes, M. and Cheng, H. Gastroenterol. 116(1):7-14. 1999; Brittan, M. and Wright, N. A. J. Pathol. 197(4):492-5. 2002), which supply the entire epithelial cell surface with a variety of epithelial cell subtypes (Brink, G. R., et al., Science, 294:2115. 2001; Burgess, D., et al. J. Cell Biol. 109(5):2139-44. 1989; Weiser, M., et al., Immnunol. Invest. 18(1-4):417-30. 1989; Quaroni, A. and Beaulieu, J. Gastroenterol., 113(4):1198-213. 1997). These specialized cells provide for an external environment-internal environment interface, ion and fluid secretion and reabsorption (Sanderson, I., et al., Gut. 38(6):853-8. 1996), antigen recognition (Hoyne, G., et al. Immunol. 80(2):204-8. 1993; Neutra, M., and Kraehenbuhl, J. Am. J. Trop. Med. Hyg. 50(5 Suppl.):10-3. 1994; Balimane, P., et al. J. Pharmacol. Toxicol. Methods. 44(1):301-12. 2000), hormone secretion (Schuerer-Maly, C., et al. Immunol. 81(1):85-91. 1994; Panja, A., et al. Clin Exp Immunol 100(2):298-305. 1995), and surface protection (Flemstrom, G. and Garner, A. Ciba Found Symp. 109:94-108. 1984; Schuerer-Maly, C., et al. Immunol. 81(1):85-91. 1994; Kindon, H., et al. Gastroenterol. 109(2):516-23. 1995; Panja, A., et al. Clin Exp Immunol 100(2):298-305. 1995; Podolsky, D. K. Gastroenterol. 32(1):122-6. 1997).
The epithelium forms upon stem cell differentiation (Brittan, M., and Wright, N. A. Gut. 53:899-910. 2004) within the intestinal tract. Stem cells are undifferentiated cells having high proliferative potential with the ability to self-renew. Stem cells may generate daughter cells that may undergo terminal differentiation into more than one distinct cell type (Morrison, S., et al. Cell 88(3):287-981997. 1997). Pluripotent (a cell that is able to differentiate into many cell types) stem cells undergo further specialization into multipotent progenitor cells that then give rise to functional cells. For example, hematopoietic stem cells give rise to red blood cells, white blood cells, and platelets. Mesenchymal stem cells are multipotent cells that are capable of differentiating along several lineage pathways, including, but not limited to, chondrocytes, osteoblasts, adipocytes, fibroblasts, marrow stroma, and other tissues of mesenchymal origin. Epithelial stem cells give rise to the various types of skin cells; and muscle satellite cells contribute to differentiated muscle tissue. The technologies for retrieval, and maintenance of such cells in an undifferentiated state, of stem cells and growing them in vitro have been the subject of study.
Molecular Markers of Gastrointestinal Epithelial Stem Cells
The surfaces of all cells in the body are coated with specialized protein receptors that have the capability to selectively bind or adhere to other signaling molecules (Weiss and Littman Cell 76.263-74.1994). These receptors and the molecules that bind to them are used for communicating with other cells and for carrying out proper cell functions in the body. Each cell type has a certain combination of receptors, or markers, on their surface that makes them distinguishable from other kinds of cells.
Stem cell markers are given short-hand names based on the molecules that bind to the corresponding stem cell surface receptors. A combination of multiple markers frequently is used to identify a particular stem cell type. Researchers often identify stem cells in shorthand by a combination of marker names reflecting the marker's presence (+) or absence (−). For example, a special type of hematopoietic stem cell from blood and bone marrow called “side population” (or “SP”) is described as (CD34−/low, c-Kit®, Sca-1+).
The following markers commonly are used by skilled artisans to identify stem cells and to characterize differentiated cell types (see http://stemcells.nih.gov/info/scireport/appendixE.asp; visited 12/28/07):
MarkerCell TypeNotesCD34Hematopoietica highly glycosylated type I transmembrane proteinstem cell (HSC),expressed on 1-4% of bone marrow cellsmuscle satellite,endothelialprogenitorCD38immature T anda type II transmembrane protein found on immature T andB cellsB cells but not most mature peripheral lymphocytesCD41platelets andthe integrin αIIb subunitmegakaryocytesCD45WBC progenitorthe leukocyte common antigen found on all cells ofhematopoietic originCD105Endothelial cellsa disulfide-linked homodimer found on endothelial cells butabsent from most T and B cellsCD133primitivea pentaspan transmembrane glycoproteinhematopoieticprogenitorsCD3T cellsa member of the T cell receptor complexCD4, CD8Mature T cellsCell-surface protein markers specific for mature Tlymphocyte (WBC subtype)CD7Early T cellsAn early T cell lineage markerCD10early T and Ba type II membrane metalloproteasecell precursorsCD13granulocytes,a type II membrane metalloproteasemonocytes andtheir precursorsCD14myelomonocytica GPI-linked protein expressed mainly on myelomonocyticlineagelineage cellsCD19B cellsa component of the B cell antigen signaling complexCD33Myelomonocytica sialic acid binding protein absent from pluripotent stemprecursorscells that appears on myelomonocytic precursors afterCD34CD38WBC lineagesA Cell-surface molecule that identifies WBC lineages.Selection of CD34+/CD38− cells allows for purification ofHSC populationsCD44MesenchymalA type of cell-adhesion molecule used to identify specifictypes of mesenchymal cellsCD56NK cellsan isoform of the neural adhesion molecule foundexclusively on natural killer (NK) cells;CD127lymphocytesthe high affinity interleukin 7 receptor expressed onlymphocytesCD138Immature Ban extracellular matrix receptor found on immature B cellscells and plasmaand plasma cellscellsGlycophorin ARBCs, embryoida sialoprotein present on human RBCs and embryoidprecursorsprecursorsCD90prothymocytesa GPI-cell anchored molecule found on prothymocyte cellsin humans-c-kitHSC, MSCCell-surface receptor on BM cell types that identifies HSCand MSC; binding by fetal calf serum (FCS) enhancesproliferation of ES cells, HSCs, MSCs, and hematopoieticprogenitor cellsFetal liverendothelialCell-surface receptor protein that identifies endothelial cellkinase-1progenitor; marker of cell-cell contacts(Flk-1)
There are no universally accepted molecular markers that identify gastrointestinal stem cells. However, several markers have been used to identify stem cells in small and large intestinal tissues. These include: β-1-integrin (Jones, R., et al. J Cell Biol 175(3):505-14. 2006), mushashi-1 (Booth, C. and Potten, C. J Clin Invest 105(11):1493-9. 2000; Potten, C., et al. Differentiation 71(1):28-41. 2003; Yen, T. and Wright, N. Stem Cell Rev 2(3):203-12. 2006), CD45 (Dekaney, C., et al. Gastroenterology 129(5):1567-80. 2005; Lynch, L., et al. J Immunol 176(9):5199-204. 2006), and cytokeratin (Raju, G. Ann Acad Med Singapore 18(3):298-301. 1989).
CD45 (also called the common leukocyte antigen, T220 and B220 in mice), is a transmembrane protein with cytoplasmic protein tyrosine phosphatase (PTP) activity. CD45 is found in hematopoietic cells except erythrocytes and platelets. It has several isoforms that can be seen in the various stages of differentiation of normal hematopoietic cells (Greaves, M., et al. Blood 61(4):628-39. 1983; Alt, F., et al. Immunol Rev 89:5-30. 1986; Thomas, M. L. Ann. Rev Immunol 7:339-69. 1989; Weiss, A. and Littman, D. Cell 76(2):263-74. 1994).
Mushashi-1 is an early developmental antigenic marker of stem cells and glial/neuronal cell precursor cells (Jones, P., et al. Cell 80(1):83-93. 1995; Kayahara, T., et al. FEBS Lett 535(1-3):131-5. 2003; Potten, C., et al. Differentiation 71(1):28-41. 2003; Asai, R., et al. Dev Growth Differ 47(8):501-10. 2005).
β-1-integrin (CD29, fibronectin receptor), is a β-subunit of a heterodimer protein member of the integrin family of proteins that are membrane receptors involved in cell adhesion and recognition (Pytela, R., et al. (1985). Cell 40(1):191-8. 1985; Fujimoto, K., et al. Gastroenterol. 123(6):1941-8. 2002; Shackleton, M., et al. Nature 439(7072):84-8. 2006).
Cytokeratins are intermediate filament proteins found in the intracytoplasmic cytoskeleton of the cells that comprise epithelial tissue. Over twenty different cytokeratin polypeptides have been identified (Franke, W., et al. Differentiation 15(1):7-25. 1979; Steinert, P., et al. Cell 42(2):411-20. 1985).
There are four main epithelial cell lineages in the gastrointestinal tract: columnar epithelial cells, goblet cells, enteroendocrine chromaffin cells, and Paneth cells. Several molecular markers have been used to identify these cells (Simon, T. and Gordon, J. Curr Opin Genet Dev 5(5):577-86. 1995).
The markers used to identify columnar epithelial cells include: intestinal alkaline phosphatase (ALP1), sucrase isomaltase (SI), sodium/glucose cotransporter (SLGT1), dipeptidyl-peptidase 4 (DPP4), and CD26. Intestinal alkaline phosphatase (E.C. 3.1.3.1) is a membrane-bound enzyme localized in the brush border of enterocytes in the human intestinal epithelium. Sucrase-isomaltase (SI, EC 3.2.1.48) is an enterocyte-specific small intestine brush-border membrane disaccharidase. Dipeptidyl-peptidase 4 (E.C. 3.4.14.5) is a membrane bound serine-type peptidase. Sodium/glucose transporter (SGLT) mediates transport of glucose into epithelial cells. SGLT belongs to the sodium/glucose cotransporter family SLCA5. Two different SGLT isoforms, SGLT1 and SGLT2, have been identified to mediate renal tubular glucose reabsorption in humans. Both of them are characterized by their different substrate affinity(Panayotova-Heiermann et al. J Biol Chem 271.10029-34.1996). SGLT1 transports glucose as well as galactose, and is expressed both in the kidney and in the intestine. CD26 is a multifunctional protein of 110 KDa strongly expressed on epithelial cells (kidney proximal tubules, intestine, and bile duct) and on several types of endothelial cells and fibroblasts and on leukocyte subsets (Kikkawa et al. Biochim Biophys Acta 1751.45-51.2005; Tokunaga et al. J Histochem Cytochem 55.735-44.2007).
The markers used to identify goblet cells include mucin 2 (MUC2) and trefoil factor 3 (TFF3) (Bergstrom et al. Infect Immun 76.796-811.2008). Mucin-2, a secreted gel-forming mucin, is the major gel-forming mucin secreted by goblet cells of the small and large intestines and the main structural component of the mucus gel. Intestinal trefoil factor 3 is a nonmucin protein and a product of fully differentiated goblet cells (Chinery, R., et al. Genomics 32(2):281-4. 1996; Ogata, Inoue et al. 1998; Itoh, H., et al. Biochem J341 (Pt 2):461-72. 1999; Yamachika, T., et al. Clin Cancer Res 8(5):1092-9. 2002; Bergstrom, K, et al. (2008). Infect Immun 76(2):796-811. 2008).
The markers used to identify enteroendocrine chromaffin cells include chromogranin A (CHGA) (Ho, S., et al. Gastroenterol. 97(2):392-404. 1989; Wimley, W., et al. Protein Sci 3(9):1362-73. 1994; Moller, P., et al. Am J Pathol 149(1):9-13. 1996; Ouellette, A. J. and Selsted, M. Faseb J 10(11):1280-9. 1996; Taupin, D., et al. Lab Invest 75(1):25-32. 1996; Turner, J. R. and Odze, R. (1996). Hum Pathol 27(1):63-9. 1996; Ronnblom, A., et al. J Intern Med 245(4):91-7 1999; Wong, W., et al. (2000). J Pathol 190(1):107-13. 2000; Andersson, N., et al. Biochem Biophys Res Commun 332(2):404-10. 2005; Stewart, C. and Hillery, S. J Clin Pathol 60(11):1284-9. 2007) and synaptophysin (SYP) (Andersson, N., et al. Biochem Biophys Res Commun 332(2):404-10. 2005). Chromogranin A (CHGA) and its derived peptides, which are stored and released from dense-core secretory granules of neuroendocrine cells, have been implicated as playing multiple roles in the endocrine, cardiovascular, and nervous systems. Synaptophysin I (SYP) is a synaptic vesicle membrane protein that is ubiquitously expressed throughout the brain without a definite synaptic function.
The markers used to identify Paneth cells include lysozyme, defensin, and matrix metallopeptidase 7 (MMP7) (Ho, S., et al. Gastroenterol. 97(2):392-404. 1989). Lysozyme (LYZ or muramidase) (E.C. 3.2.1.17) catalyzes the hydrolysis of 1,4-beta-linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in a peptidoglycan and between N-acetyl-D-glucosamine residues in chitodextrins. Defensins (DEFA1) are small peptides that are produced by leukocytes and epithelial cells. Human defensin α-1 is a 3.5-kDa, 30-amino-acid peptide that has shown effector functions in host innate immunity against some microorganisms (Semenza, G. Ann. Rev Cell Biol 2:255-313. 1986; Wimley, W., et al. Protein Sci 3(9):1362-73. 1994; Moller, P., et al. Am J Pathol 149(1):9-13. 1996; Ouellette, A. and Selsted, M. Faseb J 10(11):1280-9. 1996; Taupin, D., et al. Lab Invest 75(1):25-32. 1996; Turner, J. and Odze, R. Hum Pathol 27(1):63-9. 1996). Matrix metalloproteinases (MMPs) are an important family of metal-dependant enzymes that are responsible for the degradation of extracellular matrix components. MMPs are involved in various physiologic processes including embryogenesis and tissue remodeling. They also play a key role in invasion and metastasis of tumor cells, which require proteolysis of basal membranes and extracellular matrix (Ayabe, T., et al. J Biol Chem 277(7):5219-28. 2002; Satchell, D., et al. (2003). J Biol Chem 278(16):13838-46. 2003; Weeks, C., et al. J Biol Chem 281(39):28932-42. 2006).
The epithelial cells on the surfaces of the intestinal lumen are subjected to a wide range of assaults including microbial, chemical, and physical forces (Savage, D. C. Am J Clin Nutr 25(12):1372-9. 1972; Keren, D. F. Am J Surg Pathol. 12 Suppl 1:100-5. 1988; Ouellette, A. J. and M. E. Selsted. Faseb J. 10(11):1280-9. 1996; Neutra, M. R. Am J Physiol. 274(5 Pt 1): G785-91. 1998; Owen, R. L. Semin Immunol 11(3):157-63. 1999; Neutra, M. R., et al. Nat Immunol 2(11):1004-9. 2001; Otte, J. M. and Podolsky, D. Am J Physiol Gastrointest Liver Physiol 286(4):G613-26. 2004); thus they also may contribute to patho-physiologic impairment in diseases. Additionally, these cells are targets for inflammation, infection, and malignant transformation.
Biomarkers for Pathogenesis
Several biomarkers have been utilized as potential indicators for pathogenic processes.
SCYA (small inducible cytokine subfamily) 16 and 20 (or CCL 16 and 20) are members of a family of cytokines characterized by two adjacent cysteines (C-C) involved in immunoregulatory and inflammatory processes. They display chemotactic activity for lymphocytes and monocytes but not for neutrophils (Hieshima et al. J Biol Chem 272.5846-53.1997). CCL16 has been reported to be up-regulated selectively by IL-10 in inflammatory cell recruitment and cytokine and chemokine production during ulcerative colitis (Pannellini et al. Int J Immunopathol Pharmacol 17.171-80.2004). This chemokine significantly enhances the effector and the antigen-presenting function of macrophages and augments the cytolytic activity T cell which in turn activate caspase-8 via overexpression of TNF-alpha and Fas ligand in tumor target cells (Cappello et al. J Leukoc Biol 75.135-42.2004). Intratumoral injection of adenoviral vectors expressing CCL16 prevented metastatic spread and cured 63% of mice bearing the 4T1 mammary adenocarcinoma, a model of spontaneous metastasis (Guiducci et al. J Immunol 172.4026-36.2004). CCL20 expression in intestinal epithelial-type cells is induced by proinflammatory cytokines such as IL-1 and TNF-alpha primarily through activation of NF-kappaB (Fujiie et al. Int Immunol 13.1255-63.2001).
Retinoids, which have roles in the modulation of cell growth, differentiation, and apoptosis, are mediated by nuclear retinoic acid receptors (RARs) and retinoid X receptors (RXRs) ((Clifford et al. EMBO J 15.4142-55.1996)). Altered expression of nuclear retinoid receptors is associated with the malignant transformation of human cells (Zhao et al. Exp Cell Res 219.555-61.1995). RAR beta is the best-studied RAR subtype in the biology of retinoid effects on carcinogenesis and is the receptor subtype whose expression is frequently most decreased in lung cancer. Three different isoforms of this receptor have been reported in humans: beta1, beta2 and beta4 (Zelent et al. EMBO J 10.71-81.1991). Close investigation of the importance of the distinct functions of these isoforms in the pathogenesis of cancer identifies Beta 1 and 2 as tumor suppressors (Petty et al. J Natl Cancer Inst 97.1645-51.2005), (Soprano and Soprano J Nutr 132.3809S-13S.2002). Isoform beta1 is a fetal isoform not generally detected in normal tissues of adult humans, although it is expressed in small-cell lung cancer (Houle et al. Cancer Res 54.365-9.1994). Loss of expression of RARbeta2 is associated with esophageal squamous cell carcinomas (Ralhan et al. Int. J Cancer 118.1077-89.2006), cerebral glioma (Klein et al. Neurochirurgie 51.147-54.2005), and epidermoid lung cancer ((Houle, Rochette-Egly and Bradley Proc Natl Acad Sci USA 90.985-9.1993). It's inactivation by hypermethylation is reported in oral premalignant lesions, head and neck squamous cell carcinomas (HNSCCs) and in human colon cancer (Youssef et al. Clin Cancer Res 10.1733-42.2004).
NCAM (neural cell adhesion molecule) is an epithelial cell adhesion molecule that is found in normal colon epithelium as well as in colon tumors. Roesler et al reported that this adhesion molecule was present more often in colon cancers with a more benign course compared to clinically aggressive tumors of the colon, leading to the conclusion that this molecule might serve as a tumor suppressor in colon carcinoma (Roesler et al. Am J Surg 174.251-7.1997).
The tissue inhibitor of metalloproteinase (TIMP) gene encodes an extracellular matrix protein. It has been shown to increase cell death and growth inhibition (via delaying the G1 phase), and thus is presumed to be involved in tumor suppression (Wang et al. Cancer 112.1325-36.2008; Smith et al. Cytokine 9.770-80.1997). It originally was reported in several human cell lines including CaCo-2, a colon adenocarcinoma cell line. ((Kishnani et al. Matrix Biol 14.479-88.1995). Lee et al have demonstrated greater hypermethylation of TIMP3 in colon carcinoma compared to normal colon mucosa and adenomas (Lee et al. Lab Invest 84.884-93.2004), although a subsequent study by Xu et al showed no change in the methylation of this gene (Xu et al. World J Gastroenterol 10.3441-54.2004).
The small GTP-binding proteins of Rab family have more than 30 proteins that play important roles at defined steps of vesicular transport in protein secretion and the endocytosis pathway. Rab33B is a Golgi-specific rab protein; it plays a role in the recycling of glycosyltransferases from the Golgi to the ER. (Valsdottir et al. FEBS Lett 508.201-9.2001). No reports linking this specific protein with any cancer could be found in the literature. Rab32 is down regulated in colon cancer (Mori et al. Cancer Res 64.2434-8.2004). Rab25 mRNA also was detected in several colon carcinoma lines, including LIM1215 and HT-29 (Goldenring et al. Methods Enzymol 329.225-34.2001). Some other members of the Rab family have been reported to be upregulated and aid in the development and aggressiveness of liver and several epithelial cancers (ovarian, breast, skin) (Gebhardt et al. Am J Pathol 167.243-53.2005; Cheng et al. Nat Med 10.1251-6.2004).
SOD3 (superoxide dismutase 3), a surface bound epithelial enzyme known to protect cells from oxygen free radical damage, is underexpressed in human intestinal tumor-derived epithelial cell (HITEC) lines. In a mouse model, Gao et al have shown that a chimeric recombinant SOD2/3 reduces lung leakage by 13% in acute lung injury (Gao et al. Am J Physiol Lung Cell Mol Physiol 284.L917-25.2003).
Myb (myeloblastosis) family transcription factors, A-Myb, B-Myb, and c-Myb, also called oncoproteins, share a highly conserved DNA binding domain and bind to the same DNA sequences, but have completely different biological roles. A-Myb is regulated by the cell cycle machinery. The carboxy-terminal domain of A-Myb itself acts as a cell cycle sensor; its activity is maximal during the G1/S-transition and the S-phase of the cell cycle. (Ziebold et al. Curr Biol 7.253-60.1997). This transcription factor has been linked to the regulation of proliferation and/or differentiation of normal B cells and is overexpressed in Burkitt's lymphoma cells (Golay et al. Leuk Lymphoma 26.271-9.1997; Facchinetti et al. Biochem J 324 (Pt 3).729-36.1997).
The VCAM1 (vascular cell adhesion molecule 1) gene is a member of the Ig superfamily and encodes a cell surface sialoglycoprotein expressed by cytokine IL-6 and TNFalpha in activated endothelium (Khatib et al. Am J Pathol 167.749-59.2005). VCAM1 expression is variable in different types of carcinomas; it is detectable in colon cancers (Banner, Savas and Woda Ultrastruct Pathol 19.113-8.1995) and liver metastasis (Kitakata et al. Cancer Res 62.6682-7.2002), but not in adenocarcinoma of lung (Jiang et al. Mod Pathol 11.1189-92.1998) or esophagus (Heidemann et al. Int J Oncol 28.77-85.2006), and is downregulated during nodal metastasis in breast cancer. Madhavan and Heidmann had proposed a potential role of VCAM-1 in the development of metastasis, since they found it was strongly expressed in squamous cell carcinoma (Madhavan et al. Pathol Oncol Res 8.125-8.2002; Heidemann et al. Int J Oncol 28.77-85.2006). However Lieder's group observed a gradual decrease in expression of VCAM-1 with progressive metastatic disease (Lieder et al. Anticancer Res 25.4141-7.2005).
MSH2 (Muts (Escherichia coli) Homolog 2 (colon cancer, nonpolyposis Type 1)) is a human analog of the protein found in E. coli that plays a critical role in DNA nucleotide mismatch repair. It is well known that deletion of mismatch repair genes results in microsatellite instability (MSI), which is implicated in 15-20% of colorectal cancers (Hoops and Traber Hematol Oncol Clin North Am 11.609-33.1997; Lynch and Kaul J Natl Cancer Inst 92.511-2.2000). According to a recent study by Parc et al, microsatellite unstable tumors exhibited a better recurrence free survival than microsatellite stable tumors (Parc et al. Int J Cancer 86.60-6.2000).
Apoptosis Inhibitor 2 (API2) initially was identified in mucosa associated lymphoid tumors (MALT) and subsequently has been shown to inhibit apoptosis in a p53 mediated process (Stoffel and Le Beau Hum Hered 51.1-7.2001). Carcinogenic cells that have undergone numerous genetic mutations somehow escape apoptosis. Without being limited by theory, one likely explanation is underexpression of API2 in such cells.
Interferon induced protein 56, referred to as IFI-56K, is highly inducible by interferon gamma as well as by viral stimuli. Gene array studies have shown downregulation of this mRNA in oligodendrogliomas (Huang et al. Oncogene 23.6012-22.2004), however, large-cell lymphoma-derived cell lines show significant upregulation (Gaiser et al. J Hematother Stem Cell Res 11.423-8.2002). The literature contains a limited number of reports about the possible role of this protein.
Presenilin 2 (PSEN4; Alzheimer disease 4) has been associated with Alzheimer's disease (AD) and its expression signifies the induction and/or proliferation of an inflammatory response in AD brain (Riazanskaia et al. Mol Psychiatry 7.891-8.2002).
The GTPase Ran regulates multiple cellular functions throughout the cell cycle, including nucleocytoplasmic transport, nuclear membrane and spindle assembly (Trieselmann et al. J Cell Sci 116.4791-8.2003). A gene expression profiling study by Harousseau et al. correlated abnormal expression of RAN with rapid relapses of multiple myeloma (Harousseau, Shaughnessy and Richardson Hematology Am Soc Hematol Educ Program 237-56.2004).
The Fos gene family consists of 4 members: FOS, FOSB, FOSL1, and FOSL2. These genes encode leucine zipper proteins that dimerize with proteins of the JUN family and form the transcription factor complex AP-1. The FOS proteins function as regulators of cell proliferation, differentiation, and transformation. They have been implicated in gliomas (Debinski and Gibo Mol Cancer Res 3.237-49.2005), breast cancer (Belguise et al. Oncogene 24.1434-44.2005) and colorectal adenocarcinomas (Wang et al. Int J Cancer 101.301-10.2002).
Gastrointestinal (GI) diseases, such as colon cancer, inflammatory bowel disease (IBD), short bowel syndrome, gastroesophageal reflux disease (GERD), irritable bowel syndrome (IBS), and iatrogenic injuries to the billiary epithelium, have a significant health and economic impact worldwide. Many treatments focus on symptom relief, and are not curative. Studies have suggested the main affected cellular component of these diseases is the epithelium.
Colorectal cancer (CRC) is the second leading cause of cancer death and is the third most common cause of malignancy in the U.S. While early stage disease can be cured with multimodality therapies, the majority of patients present with Stage III or IV disease (Jemal et al. CA Cancer J Clin 58.71-96.2008). The prognosis for patients with advanced CRC generally is poor.
Currently, no specific biomarker exists to aid in early diagnosis and/or management of CRC. While the actual cause of CRC remains unknown, the source of this disease is transformation of the epithelial cells lining the colon and or rectum. Previous studies with colonic tumor epithelial cell lines and/or tissues indicate that in most cases, tumor development is associated with aberrant gene expression (Rieker et al. Pathol Oncol Res 14.199-204.2008; Hagymasi et al. Ory Hetil 148.779-85.2007). Comparisons of the gene and/or protein expression profiles of normal and cancerous tissues in histological samples have identified several candidates that may be responsible for cancer development (Solmi et al. BMC Cancer 6.250.2006; Lepourcelet et al. Development 132.415-27.2005; Solmi et al. Int J Oncol 25.1049-56.2004; Ohnishi et al. Cancer Res 58.2440-4.1998; Hargest and Williamson Gut 37.826-9.1995). However, none of these genes or molecules have proved to be specific. This partially may be due to the fact that studies that compare differential gene expression between tumor and normal colorectal epithelium have been limited by the lack of paired normal and tumor-derived colorectal epithelial cell lines from the same individual.
Identification of early stage CRC biomarkers is vital for earlier CRC detection, biopsies, therapeutic target identification, drug testing, efficacy and treatment. However, significant difficulties for identifying these biomarkers exist. Most subjects with early CRC are asymptomatic, and symptoms usually do not appear until the cancer has reached an advanced stage (Schneider et al. Cancer 110.2075-82.2007; Glimelius et al. Acta Oncol 31.645-51.1992). Additionally, many individuals do not avail themselves of colonoscopy (the best means of early detection) due to its costly and invasive nature. Therefore, a high percentage of CRC cases are not detected until the cancer has progressed substantially, invaded adjoining tissues, and/or metastasized, which accounts for both the high percentage of reoccurrence and relatively high mortality rate observed (Walgenbach-Brunagel et al. J Cell Biochem 104.286-94.2008; Shapero et al. Gastrointest Endosc 65.640-5.2007). Further, the vast majority of CRC biomarkers that have been discovered do not have a function in vivo, are not very effective in early detection of CRC, recognize only a small subset of cancers, and often only detect CRC after it has progressed to later stages and grades.
Research efforts have utilized whole tissue sections, peripheral blood cells, serum and urine. These models do not allow the identification of the specific cellular, molecular, and genetic changes that the colonic epithelium undergoes throughout its malignant transformation.
Chronic inflammatory diseases, such as, for example, ulcerative colitis (UC) and Crohn's disease (CD), have been known for many years to predispose to cancer development (Herszenyi, Miheller and Tulassay Dig Dis 25.267-9.2007; Svrcek et al. Histopathology 50.574-83.2007; van Hogezand et al. Scand J Gastroenterol Suppl 48-53.2002).
Models incorporating malignant transformed cell lines, isolated epithelial cells, and animals have been used to study the functional capabilities, and alterations in the acute and chronic inflammatory or malignant states of epithelial cells. However, each of these model systems has limitations and differs markedly from an in vivo system of primary epithelial cells. For example, malignant cell lines usually have chromosomal abnormalities that cause instability. Tumor lines also differ from cell line to cell line, and from passage to passage (Jobin et al. J Immunol 158.226-34.1997; Khan et al. Anticancer Res 11.1343-8.1991; Owen-Schaub et al. Cancer Res 54.1580-6.1994). The use of freshly isolated epithelial cells often is complicated due to the mixture of several cell types present (T cells, B cells, or macrophages), especially when diseased tissue is used as cell source). Additionally, the isolation procedure can alter the phenotype of the cells by cleaving certain molecules from the cell surface. Furthermore, surface and crypt epithelial cells may have different phenotypes and functions and are not usually used in fully separated populations.
A model incorporating SV40 transformed epithelial cell lines established from human, mouse and rat epithelium (Brandsch et al. Scand J Gastroenterol 33.833-8.1998; Moyer et al. Prog Clin Biol Res 279.363-72.1988; Quaroni and Beaulieu Gastroenterology 113.1198-213.1997; Schorkhuber et al. Cell Biol Toxicol 14.211-23.1998). has been utilized. Although this model system allows the cells to retain some specific functions and to survive indefinitely, there has been concern about the altered growth control in these cells through the transfection of viral DNA (such as the large T—antigen gene from SV40) (Hauft et al. J Cell Biol 117.825-39.1992; Kim et al. Dev Biol Stand 94.297-302.1998; Ozer Prog Mol Subcell Biol 24.121-53.2000). Similarly, findings with animal model systems show that these do not always mimic precisely the functional scenarios of human intestinal epithelial cells in vivo (Seshimo et al. Cell Struct Funct 18.345-54.1993).
Cell lines of non-transformed human intestinal epithelial cells and of colorectal cancer cells (derived from primary cells and not artificially transformed) have long been needed for advancing research into the cause(s), prevention, treatment, and cure of colorectal cancer as well as other GI disorders such as, but not limited to, inflammatory bowel disease (IBD), Barrett's esophagitis/esophageal adenocarcinoma, gastritis, gastric cancer, ulcerative colitis (UC), Crohn's Disease (CD), and irritable bowel syndrome.
An understanding of the pathogenesis of CRC is heavily dependent on identifying differences between normal and tumorous colon epithelium. A non-transformed comparative cell line model using cells derived from normal and tumorous colon epithelium from the same individual would provide insight into these differences. However, no such model has been available. Therefore a need exists for an adequate preclinical model that provides an environment similar to the physiological environment of a human gastrointestinal tract.
The described invention provides a preclinical, in vitro system comprising gastrointestinal epithelial stem cell-like progenitor cells having the structural and functional characteristics of the normal human gastrointestinal tract. The system is useful for identifying specific bio-molecules that are involved in, or indicative of, inflammatory (e.g. ulcerative colitis and Crohn's disease) processes and/or cancerous development. Further, the specific biomolecules involved in, or indicative of, cancerous development may be used to develop and commercialize diagnostic and/or therapeutic constructs (such as antibodies, peptides, and small interfering RNA) against these molecules.
The described invention also provides a system useful for simulating microenvironments that may cause malignant changes on normal Human Intestinal Primary Epithelial Cells (HIPECs) as well as for evaluating of the effects of various therapeutic agents in reversing the malignant transformation process.
The described invention further provides a cell system useful for identifying biomarkers for colon cancer and other tumors of the gastrointestinal tract. The described invention moreover provides a system useful for studying differential alterations in the cellular machinery of tumorous epithelial stem cells by comparing these cells with their normal counterparts.